The rapid global acceptance of intracytoplasmic sperm injection (ICSI) therapy in infertility clinics as advanced far faster than the fundamental knowledge regarding its molecular and cell biological foundations. However, it is not yet possible to absolutely conclude that there are no long-lasting and unanticipated consequences of assisted reproductive technology (ART) using ICSI. Since the deliberate creation of human zygotes for biomedical research is unlikely to ever be free of religious, ethical,moral, political and financial complexities, the goal of this project is to identify the variations between ICSI and natural fertilization, and to provide the essential scientific data to understand the molecular and cell biological basis of this approach in a clinically relevant system. Since ICSI differs from natural fertilization in several ways, the overall objective of this project is to provide a complete evaluation of the consequences of various oocyte and sperm manipulations during ICSI in terms of subsequent embryo development and the production of offspring. To donors of varying fertility and from rhesus monkeys, as well as oocytes from the non-human primate and rabbit. Human oocytes, discarded as unfertilized or failures after ICSI, will be investigated using non-federal funding. Aim 1. To develop a clinically relevant system for exploring the mechanisms and safety of ICSI; Aim 2. TO investigate egg activation, first cell cycle progression and the fates of sperm components after ICSI; and Aim 3. To develop a heterologous system (human sperm microinjected into rabbit oocytes) to assay human sperm quality. We propose to perform clinical and preclinical studies using gametes and embryos obtained from monkeys, and donated, clinically discarded human specimens from informed consenting patients. Taken together, this information will advance: clinically-relevant knowledge about genomic union during ICSI; may translate into applications for the diagnosis of male infertility; and may well inform and reassure infertile patients and their physicians, about the safety and biomedical basis of this powerful, but still experimental, therapeutic approach.